Hybridomas producing Clostridium botulinum, an anaerobic spore-forming bacterium, produces a family of botulinum neurotoxins (BoNT, EC 3.4.24.69) [Gill, M. Microbiol. Rev. 46:86-94 (1982)] consisting of seven serotypes, A-G (BoNT/A-BoNT/G). These are considered the most toxic proteins known. Serotype A is synthesized as a single 1,296 amino acid polypeptide, ˜150,000 Dalton (Da) that is then cleaved endogenously or exogenously forming a dichain molecule comprised of an ˜100 kDa heavy chain (HC) and an ˜50 kDa light chain (LC) linked by a single disulfide bond Montecucco, C, and Schiavo, G. Structure and function of tetanus and botulinum neurotoxins. Quarterly Rev. Biophys. 28:423-472 (1995)]. The HC mediates toxin entry into neurons, and the LC functions as a zinc-dependent endoprotease cleaving SNAR proteins involved in acetylcholine release resulting in muscular paralysis [Turton, K., Chaddock, J. A., Acharya, K. R. Trends Biochem. Sci. 27:552-558 (2002)]. The crystal structure of BoNT/A was determined at 3.3 Å resolution [Lacyt, D. B., Tepp, W., Cohen, A. C., DasGupta, B. R., and Stevens, R. C. Crystal structure of botulinum neurotoxin type A and implications for toxicity Nature Structural Biol. 5:898-902 (1998)].
BoNT is synthesized as a single 150 kDa precursor protein, which is cleaved to form two subunit polypeptides, linked by a single disulfide bond. The gold standard for BoNT detection is the mouse bioassay. The mouse bioassay is time consuming, up to 4 days, and lacks specificity, it has a sensitivity in the low pictogram range. Most BoNT immunoassays reported appear to have much less sensitivity than the mouse bioassay.
Bavari et al., U.S. Pat. Nos. 6,667,158 and 7,049,085 have disclosed antibodies to BoNT/A and associated methods of use wherein the affinity of the